Lag-3 antibody, antigen-binding fragment thereof, and pharmaceutical application thereof

ABSTRACT

Provided are a LAG-3 antibody, an antigen-binding fragment thereof, and a pharmaceutical application thereof. Further, provided are a chimeric antibody comprising a CDR of the LAG-3 antibody, a humanized antibody, a pharmaceutical composition comprising the LAG-3 antibody and the antigen-binding fragment thereof, and an application of the pharmaceutical composition as an antineoplastic drug. Particularly, provided is an application of a humanized LAG-3 antibody in preparation of drugs for treatment of diseases involving immune cells.

FIELD OF THE INVENTION

The present invention relates to a LAG-3 antibody, antigen-bindingfragment thereof, a chimeric antibody or a humanized antibody comprisingthe CDR regions of the LAG-3 antibody, as well as pharmaceuticalcompositions comprising the LAG-3 antibody and the antigen-bindingfragment thereof, as well as its use as an anti-cancer drug.

BACKGROUND OF THE INVENTION

Lymphocyte Activation Gene-3, also known as LAG-3 or CD223, is a memberof the immunoglobulin superfamily, which can negatively regulate variousfunctions and survival cycles of immune cells. Studies have shown thatLAG-3 plays an important role in viral infection, autoimmune diseasesand tumor-induced immune system dysfunction. Influencing the function ofLAG-3 can improve the status of immune dysfunction during thedevelopment of these diseases, so as to improve the prognosis of thediseases.

As a member of the immunoglobulin superfamily, LAG-3 is composed ofthree regions: extracellular domain, transmembrane region and thecytoplasmic domain. The mature LAG-3 molecule, which was firstdiscovered by Triebel et al. in 1990 (J Exp Med, 1990, 171 (5):1393-405), consists of 470 amino acids with a relative molecular weightof 70 kDa. It has been found that LAG-3, like CTLA-4 and PD-1, is anegative co-stimulatory molecule, the activation of which can negativelyregulate function of lymphocyte. Structurally, LAG-3 is closely relatedto CD4, but it has reverse function to CD4. For example, LAG-3 moleculehas high similarity to CD4 molecule, and both can bind to MHC-II (MajorHistocompatibility Complex) class molecules. However, the bindingavidity of LAG-3 to MHC-II molecules is higher than that of CD4. Thus,it intervenes in TCR activation induced by CD4⁺T lymphocyte cells andinhibits the activation of T lymphocyte (Curr Opin Immunol, 2009,21(2):179-86; Eur J Immunol, 2003, 33 (4): 970-9). In vitro studies, ithas been shown that LAG-3 can inhibit the proliferation of T lymphocyteinduced by antigen. Blocking LAG-3 will improve activation andproliferation of T lymphocyte, and improve the cytokines secreted bytype 1 T helper cells (Th1). Huang et al. have showed that the level ofLAG-3 on the activated CD4+ Treg cells was significantly increased, andLAG-3 was a necessary condition for CD4⁺ Tregs to exert the greatestimmunosuppressive effect (Immunity, 2004, 21 (4): 503-13). In addition,anti-LAG-3 antibody also maintains the homeostasis of CD4⁺ and CD8⁺Tlymphocyte, blocking LAG-3 will significantly enhance the ability ofCD8⁺T lymphocytes to kill tumor cells (J Clin Invest, 2007, 117 (11):3383-92). Some studies on diseases have also indicated that LAG-3 playsan important role in the regulating development and progression of adisease. Gandhi et al. verified that the expression level of LAG-3 in Tlymphocytes of human lymphoma tissue is associated with T lymphocytedysfunction, and clearance of LAG-3⁺T lymphocytes can significantlyenhance the ability of eliminating tumor cell by T lymphocytes (Blood,2006, 108 (7): 2280-9). The results show that LAG-3 is an importantinhibitory molecule on the surface of immune cells and has a significantnegative regulatory effect on T lymphocytes.

LAG-3 is mainly expressed on T lymphocytes, B lymphocytes, NK cells,Treg cells and DC cells (Proc Natl Acad Sci USA, 1997, 94 (11): 5744-9.Eur J Immunol, 2005, 35 (7): 2081-8; J Immunol, 2009, 182 (4): 1885-91).LAG-3 is a class of immunosuppressive molecules, and is one of thecomponents constituting the co-receptor of TCR. It intervenes in TCRactivation induced by T lymphocyte, and plays a negatively regulatoryrole in the activation of T lymphocytes. In some diseases, theexpression of LAG-3 was increased, and the correspondingimmunosuppression was observed. Gandhi et al. found that the lymphocytesin the blood and tumor tissues from patients with Hoggkin's lymphomahighly expressed LAG-3; and the function of specific CD8⁺T cells wasobviously impaired in tumor tissues, if the LAG-3-positive T cell wasremoved, the anti-tumor function was restored and cytokine secretion wasincreased. It was speculated that the expression of LAG-3 is associatedwith the negative regulation of the immune function of specific T cells,inhibiting the function of LAG-3 molecule can enhance the anti-tumoreffect of T cell, so that LAG-3 molecule may be a potential target fortumor immunotherapy (Blood, 2006, 108 (7): 2280-9).

Currently there are several multinational pharmaceutical companies, suchas BMS and Novartis, engaging in the study of monoclonal antibodiesagainst LAG-3, which enhance the anti-tumor effect of T cells andmaximize the patients' own immune response to the tumor by stimulatingantigen-specific T cell responses, and subsequently achieve the purposeto kill tumor cells. The currently relating patents are, such asWO2010019570, WO2014008218, WO9530750, WO2004078928, WO2008132601,WO2014140180 and WO2015138920.

The present invention provides a LAG-3 antibody with high affinity, highselectivity, and high biological activity.

SUMMARY OF THE INVENTION

The present invention provides a LAG-3 antibody or an antigen-bindingfragment thereof, comprising any one or more of the CDR region selectedfrom the following (i) or (ii) or sequences with at least 85%identity(preferably 95%) to the following:

(i) HCDR regions as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11; and LCDR regions as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ IDNO: 17; or

(ii) HCDR regions as shown in: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ IDNO: 14; and LCDR regions as shown in SEQ ID NO: 18, SEQ ID NO: 19 andSEQ ID NO: 20.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the heavy chain variable region comprisesHCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ IDNO: 11, respectively, or sequences with at least 85% (preferably 95%)identity to these sequences.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or antigen-binding fragment thereof according to thepresent invention, wherein the heavy chain variable region comprisesHCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQID NO: 14, respectively, or sequences with at least 85% (preferably 95%)identity to these sequences.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the light chain variable region comprisesLCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQID NO: 17, respectively, or sequences with at least 85% (preferably 95%)identity to these sequences;

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the light chain variable region comprisesLCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQID NO: 20, respectively, or sequences with at least 85% (preferably 95%)identity to these sequences.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the antibody or the antigen-binding fragmentthereof is a murine antibody or a fragment thereof.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the antibody light chain variable regionfurther comprises light chain FR region derived from murine κ chain or avariant thereof, or light chain FR region derived from murine λ chain ora variant thereof; wherein the antibody heavy chain variable regionfurther comprises heavy chain FR region derived from murine IgG1 or avariant thereof, or heavy chain FR region derived from murine IgG2 or avariant thereof, or heavy chain FR region derived from murine IgG3 or avariant thereof.

In another preferred embodiment of the present invention, provided amurine LAG-3 antibody or antigen-binding fragment thereof according tothe present invention, wherein the murine antibody comprises a heavychain variable region as shown in SEQ ID NO: 5 and a light chainvariable region as shown in SEQ ID NO: 6.

In another preferred embodiment of the present invention, provided amurine LAG-3 antibody or the antigen-binding fragment thereof accordingto the present invention, wherein the murine antibody comprises a heavychain variable region as shown in SEQ ID NO: 7 and a light chainvariable region as shown in SEQ ID NO: 8.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the antibody light chain further compriseslight chain constant region derived from murine κ chain or a variantthereof, or light chain constant region derived from murine λ chain or avariant thereof; wherein the antibody heavy chain variable regionfurther comprises heavy chain FR region derived from murine IgG1 or avariant thereof, or heavy chain FR region derived from murine IgG2 or avariant thereof, or heavy chain FR region derived from murine IgG3 or avariant thereof.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or antigen-binding fragment thereof according to thepresent invention, wherein the antibody or the antigen-binding fragmentthereof is a chimeric antibody or a fragment thereof.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or antigen-binding fragment thereof according to thepresent invention, wherein the antibody or the antigen-binding fragmentthereof is a humanized antibody or a fragment thereof.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the heavy chain FR sequence of the heavychain variable region of the humanized antibody is derived from acombination sequence of human germline heavy chain IGHV7-4-1*02 andhjh6.1, or derived from mutant sequence thereof; It comprises FR1, FR2,FR3 from human germline heavy chain IGHV7-4-1*02 and FR4 from hjh6.1, orthe mutant sequence thereof; preferably, the heavy chain FR sequence ofthe humanized antibody has 0-10 amino acid back-mutations, morepreferably has one or more back-mutations selected from the groupconsisting of E46K, R38K, V93T and Y95F.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or antigen-binding fragment thereof according to thepresent invention, wherein the humanized antibody heavy chain variableregion sequence is as shown in SEQ ID NO: 21, or a sequence with atleast 85% (preferably 95%) identity to the sequence; preferably thereare 1-10 amino acid changes in the heavy chain variable region. Theseamino acid changes may be made based on technology of affinitymaturation in the art.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein humanized antibody heavy chain variableregion comprises the sequence of SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 24 or SEQ ID NO: 25, or a sequence with at least 85% (preferably95%) identity to these sequences.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the heavy chain FR sequence of the heavychain variable region of the humanized antibody is derived from acombination sequence of human germline heavy chain IGHV1-3*01 andhjh6.1, or mutant sequences thereof; preferably it comprises FR1, FR2,FR3 from human germline heavy chain IGHV1-3*01 and FR4 from hjh6.1, orthe mutant sequence thereof; wherein the heavy chain FR sequence of thehumanized antibody has 0-10 amino acid back-mutations, more preferablyone or more back-mutations selected from the group consisting of F29L,A97T, M481, V68A, 170L, R72V and T74K.

In another preferred embodiment of the present invention, provided ahumanized LAG-3 antibody or antigen-binding fragments thereof accordingto the present invention, wherein the humanized antibody heavy chainvariable region sequence is as shown in SEQ ID NO: 29, or a sequencewith at least 85% (preferably 95%) identity to the above sequence;preferably there are 1-10 amino acid changes in the heavy chain variableregion; These amino acid changes may be made based on technology ofaffinity maturation in the art.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the heavy chain variableregion sequence of the humanized antibody is selected from the sequencesof SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33, or asequence with at least 85% (preferably 95%) identity to these sequences.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain FR sequenceof the humanized antibody light chain variable region is derived from acombination sequence of human germline light chain IGKV1-39*01 andhjk4.1 and the mutant sequences thereof, it comprises FR1, FR2, FR3 fromhuman germline light chain IGKV1-39*01, and FR4 from hjk4.1 and themutant sequence thereof.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain variableregion sequence of the humanized antibody is as shown in SEQ ID NO: 22,or a sequence with at least 85% identity to this sequence; preferablythere are 1-10 amino acid changes in the light chain variable region.These amino acid changes may be made based on technology of affinitymaturation in the art.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain FR sequenceof the humanized antibody has 0-10 amino acid back-mutations, preferablyhas one or more back-mutations selected from the group consisting ofD70Q, F71Y, I48V and A43S.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain variableregion sequence of the humanized antibody is selected from sequence ofSEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, or asequence with at least 85% (preferably 95%) identity to these sequences.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain variableregion sequence of the humanized antibody is shown in SEQ ID NO: 30, ora sequence with at least 85% identity to this sequence; preferably thereare 0-10 amino acid changes in the light chain variable region; Theseamino acid changes may be made based on technology of affinitymaturation in the art.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain FR sequenceof the humanized antibody has 0-10 amino acid back-mutations, preferablyhas one or more back-mutations selected from the group consisting ofL46R, G66R, S60K, P44F, Y36L, K42G I21L and T85D.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the light chain variableregion sequence of the humanized antibody is selected from sequence ofSEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO:37, or a sequence with at least 85% (preferably 95%) identity to thesesequences.

In another preferred embodiment of the present invention, provided thehumanized LAG-3 antibody or the antigen-binding fragment thereofaccording to the present invention, wherein the humanized antibodycomprises:

(a) heavy chain variable region sequence, wherein the heavy chainvariable region sequence has at least 85% (preferably 95%) identity tothe sequence selected from the group consisting of SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 24 and SEQ ID NO: 25; and

(b) light chain variable region sequence, wherein the light chainvariable region sequence has at least 85% identity to sequence selectedfrom the group consisting of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27and SEQ ID NO: 28.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the humanized antibody comprises:

(a) heavy chain variable region sequence, wherein the heavy chainvariable region sequence has at least 85% (preferably 95%) identity tothe sequence selected from the group consisting of SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, and

(b) light chain variable region sequence, wherein the light chainvariable region sequence has at least 85% (preferably 95%) identity tothe sequence selected from the group consisting of SEQ ID NO: 30, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the antibody comprises a combination of aheavy chain variable region and a light chain variable region selectedfrom the group consisting of:

1) the heavy chain variable region of SEQ ID NO: 21 and the light chainvariable region of SEQ ID NO: 22;

2) the heavy chain variable region of SEQ ID NO: 21 and the light chainvariable region of SEQ ID NO: 26;

3) the heavy chain variable region of SEQ ID NO: 21 and the light chainvariable region of SEQ ID NO: 27;

4) the heavy chain variable region of SEQ ID NO: 21 and the light chainvariable region of SEQ ID NO: 28;

5) the heavy chain variable region of SEQ ID NO: 23 and the light chainvariable region of SEQ ID NO: 22;

6) the heavy chain variable region of SEQ ID NO: 23 and the light chainvariable region of SEQ ID NO: 26;

7) the heavy chain variable region of SEQ ID NO: 23 and the light chainvariable region of SEQ ID NO: 27;

8) the heavy chain variable region of SEQ ID NO: 23 and the light chainvariable region of SEQ ID NO: 28;

9) the heavy chain variable region of SEQ ID NO: 24 and the light chainvariable region of SEQ ID NO: 22;

10) the heavy chain variable region of SEQ ID NO: 24 and the light chainvariable region of SEQ ID NO: 26;

11) the heavy chain variable region of SEQ ID NO: 24 and the light chainvariable region of SEQ ID NO: 27;

12) the heavy chain variable region of SEQ ID NO: 24 and the light chainvariable region of SEQ ID NO: 28;

13) the heavy chain variable region of SEQ ID NO: 25 and the light chainvariable region of SEQ ID NO: 22;

14) the heavy chain variable region of SEQ ID NO: 25 and the light chainvariable region of SEQ ID NO: 26;

15) the heavy chain variable region of SEQ ID NO: 25 and the light chainvariable region of SEQ ID NO: 27; and 16) the heavy chain variableregion of SEQ ID NO: 25 and the light chain variable region of SEQ IDNO: 28.

In another preferred embodiment of the present invention, provided theLAG-3 antibody or the antigen-binding fragment thereof according to thepresent invention, wherein the antibody comprises a combination of aheavy chain variable region and a light chain variable region selectedfrom the group consisting of:

1) the heavy chain variable region of SEQ ID NO: 29 and the light chainvariable region of SEQ ID NO: 30;

2) the heavy chain variable region of SEQ ID NO: 29 and the light chainvariable region of SEQ ID NO: 34;

3) the heavy chain variable region of SEQ ID NO: 29 and the light chainvariable region of SEQ ID NO: 35;

4) the heavy chain variable region of SEQ ID NO: 29 and the light chainvariable region of SEQ ID NO: 36;

5) the heavy chain variable region of SEQ ID NO: 29 and the light chainvariable region of SEQ ID NO: 37;

6) the heavy chain variable region of SEQ ID NO: 31 and the light chainvariable region of SEQ ID NO: 30;

7) the heavy chain variable region of SEQ ID NO: 31 and the light chainvariable region of SEQ ID NO: 34;

8) the heavy chain variable region of SEQ ID NO: 31 and the light chainvariable region of SEQ ID NO: 35;

9) the heavy chain variable region of SEQ ID NO: 31 and the light chainvariable region of SEQ ID NO: 36;

10) the heavy chain variable region of SEQ ID NO: 31 and the light chainvariable region of SEQ ID NO: 37;

11) the heavy chain variable region of SEQ ID NO: 32 and the light chainvariable region of SEQ ID NO: 30;

12) the heavy chain variable region of SEQ ID NO: 32 and the light chainvariable region of SEQ ID NO: 34;

13) the heavy chain variable region of SEQ ID NO: 32 and the light chainvariable region of SEQ ID NO: 35;

14) the heavy chain variable region of SEQ ID NO: 32 and the light chainvariable region of SEQ ID NO: 36;

15) the heavy chain variable region of SEQ ID NO: 32 and the light chainvariable region sequence of SEQ ID NO: 37;

16) the heavy chain variable region of SEQ ID NO: 33 and the light chainvariable region of SEQ ID NO: 30;

17) the heavy chain variable region of SEQ ID NO: 33 and the light chainvariable region of SEQ ID NO: 34;

18) the heavy chain variable region of SEQ ID NO: 33 and the light chainvariable region of SEQ ID NO: 35;

19) the heavy chain variable region of SEQ ID NO: 33 and the light chainvariable region of SEQ ID NO: 36; and

20) the heavy chain variable region of SEQ ID NO: 33 and the light chainvariable region of SEQ ID NO: 37.

In another preferred embodiment of the present invention, provided achimeric or humanized LAG-3 antibody or the antigen-binding fragmentthereof according to the present invention, wherein the heavy chain ofthe chimeric antibody or the humanized antibody further comprises aheavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4or a variant thereof, preferably comprises a heavy chain constant regionderived from human IgG4 or a variant thereof, most preferably comprisesa heavy chain constant region as shown in SEQ ID NO: 38.

The light chain of said chimeric antibody or said humanized antibodyfurther comprises light chain constant region derived from human κchain, human λ chain or a variant thereof, most preferably comprises alight chain constant region as shown in SEQ ID NO: 39.

The present invention further provides a pharmaceutical compositionwhich comprises a therapeutically effective amount of the LAG-3 antibodyor the antigen-binging fragment thereof described herein and one or morepharmaceutically acceptable carriers, diluents or excipients.

The invention further provides an isolated monoclonal antibody orantigen-binding fragment thereof which competes for binding to LAG-3with the monoclonal antibody or antigen-binding fragment thereof asdescribed above.

The present invention further provides an isolated nucleic acid encodingthe LAG-3 antibody or the antigen-binding fragment described above.

The present invention further provides an expression vector comprisingthe isolated nucleic acid as described above.

The present invention further provides a host cell transformed with theexpression vector as described above, wherein the host cell is selectedfrom the group consisting of prokaryotic cells and eukaryotic cells,preferably eukaryotic cells, more preferably mammalian cells.

The present invention further provides a method for preparing a LAG-3antibody or the antigen-binding fragment thereof, comprising expressingthe antibody or the antigen-binding fragment thereof in the host cell asdescribed above and isolating the antibody or the antigen-bindingfragment thereof from the host cell.

The present invention further provides a method for inhibiting thegrowth of tumor cells in a subject, comprising administering to thesubject a therapeutically effective amount of the LAG-3 antibody or theantigen-binding fragment thereof according to the invention, or thepharmaceutical composition comprising the same, thereby inhibiting thetumor growth in the subject.

The present invention further provides use of the above LAG-3 antibodyor the antigen-binding fragment thereof, or the pharmaceuticalcomposition containing the same, in the inhibition of tumor cell growthin a subject.

The present invention further provides use of said LAG-3 antibody or theantigen-binding fragment thereof, or the pharmaceutical compositioncontaining the same, in the preparation of a medicament for inhibitingthe growth of tumor cells in a subject.

The present invention further provides use of the LAG-3 antibody or theantigen-binding fragment thereof according to the present invention, orthe pharmaceutical composition comprising the same, or the nucleic aciddescribed above, in the preparation of a medicament for the treatment ofa disease or a condition associated with the involvement of immunecells, wherein the disease or the condition is preferably a cancer. Thecancer described herein includes but not limited to ovarian cancer,melanoma (for example, metastatic malignant melanoma), prostate cancer,intestinal cancer (for example, colon and small intestinal cancer),stomach cancer, esophageal cancer, breast cancer, lung cancer, renalcancer (for example, clear cell carcinoma), pancreatic cancer, uterinecancer, liver cancer, bladder cancer, cervical cancer, oral cancer,brain cancer, testicular cancer, skin cancer, thyroid cancer, andhematological malignant tumors including myeloma and chronic/acuteleukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Humanized anti-LAG-3 antibody enhances secretion of IL-2cytokine from T lymphocytes activated by SEB. The results show thathumanized LAG-3 antibody candidates, Hu229-013 and Hu303-005, canenhance the secretion of cytokine IL-2 from the activated T lymphocytes,and with dose-effect of drug concentration.

FIG. 2: Effect of humanized anti-LAG-3 antibody on tumor volume ofU-87MG tumor-bearing mice. The results show that both LAG-3 antibodyHu229-013 6mpk and Hu303-005 6mpk have certain anti-tumor effects, andthe tumor inhibition rates were 27.25% (p<0.05) and 34.94% (p<0.01),respectively, and there were significant differences compared to thecontrol group (p<0.001 vs hIGg).

DETAILED DESCRIPTION OF THE INVENTION 1. Terms

In order to more readily understand the invention, certain technical andscientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, the single-letter code and the three-letter code foramino acids are as described in J. Biol. Chem, 243, (1968) p3558.

The term “LAG-3” refers to Lymphocyte Activation Gene-3. The term“LAG-3” includes variants, isoforms, homologs, orthologs and paralogs.The term “human LAG-3” refers to the sequence of human LAG-3, such asthe complete amino acid sequence of human LAG-3 with Uniprot No. P18627.LAG-3 is also known as in the art, for example, CD223. The human LAG-3sequence may differ from human LAG-3 of Uniprot No. P18627 in that,e.g., the human LAG-3 has conserved mutations or mutations innon-conserved regions and it has substantially the same biologicalfunction as the human LAG-3 of Uniprot No. P18627. For example, abiological function of human LAG-3 is an epitope in the extracellulardomain of LAG-3 that is specifically bound by the antibody disclosedherein, or a biological function of human LAG-3 is binding to MHC ClassII molecules.

A particular human LAG-3 sequence will generally have at least 90%identity in amino acid sequence to human LAG-3 of Uniprot No. P18627 andcontains amino acid residues which are identified as being human aminoacid sequences when compared to LAG-3 amino acid sequences from otherspecies (e.g., murine). In certain cases, a human LAG-3 can have atleast 85%, or even at least 95%, 96%, 97%, 98%, or 99% identity in aminoacid sequence to LAG-3 of Uniprot No. P18627. In certain embodiments, ahuman LAG-3 sequence will display no more than 10 amino acid differencesfrom the LAG-3 sequence of Uniprot No. P18627. In certain embodiments,the human LAG-3 can display no more than 5, or even no more than 4, 3,2, or 1 amino acid difference from the LAG-3 sequence of Uniprot No.P18627. Percent identity can be determined as described herein.

As used herein, “Sequence identity” indicates the degree of identitybetween two nucleic acids or two amino acid sequences when optimallyaligned and compared in the case of having mutations such as appropriatesubstitutions, insertions or deletions. The sequence identity betweenthe sequence described in the present invention and the correspondingsequence is at least 85%, 90% or 95%, preferably at least 95%.Representative examples include, but are not limited to, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical positions/total number of positions multiplied by 100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using the default settings ofthe BLASTN/BLASTP algorithm available on the National Center forBiotechnology Institute's website.

As used herein, “Antibody” refers to immunoglobulin, a four-peptidechain structure connected together by disulfide bonds between twoidentity heavy chains and two identity light chains. Differentimmunoglobulin heavy chain constant regions exhibit different amino acidcompositions and rank orders, hence present different kinds ofantigenicity. Accordingly, immunoglobulins can be divided into fivecategories, or called as immunoglobulin isotypes, namely IgM, IgD, IgG,IgA and IgE, their heavy chains are μ chain, δ chain, γ chain, α chainand ε chain, respectively. According to its amino acid composition ofhinge region and the number and location of heavy chain disulfide bonds,the same type of Ig can be divided into different sub-categories, forexample, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chaincan be divided into κ or λ chain considering of different constantregion. Each of the five types of IgG can have κ or λ chain.

In the present invention, the antibody light chain mentioned hereinfurther comprises a light chain constant region, which comprises a humanor murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain mentioned hereinfurther comprises a heavy chain constant region, which comprises humanor murine IgG1, IgG 2, IgG 3, IgG 4 or a variant thereof.

Near the N-terminal sequence of the antibody heavy and light chains,about 110 of amino acids change largely, known as variable region (Fvregion); the rest of the amino acid sequence near the C-terminus isrelative stable, known as constant region. Variable region comprisesthree hypervariable regions (HVR) and four relatively conserved sequenceframework regions (FR). Three hypervariable regions determine thespecificity of the antibody, also known as complementarity determiningregion (CDR). Each light chain variable region (LCVR) and each heavychain variable region (HCVR) is composed of three CDR regions and fourFR regions, with sequential order from the amino terminus to thecarboxyl terminus being: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Threelight chain CDRs refer to LCDR1, LCDR2, and LCDR3; three heavy chainCDRs refer to HCDR1, HCDR2 and HCDR3. The number and location of CDRregion amino acid residues in LCVR and HCVR regions of the antibody orantigen binding fragment herein comply with known Kabat numberingcriteria (LCDR1-3, HCDE2-3), or comply with Kabat and Chothia numberingcriteria (HCDR1).

The antibody of the present invention comprises murine antibody,chimeric antibody and humanized antibody, preferable humanized antibody.

The term “murine antibody” in the present invention refers to anti-humanLAG-3 monoclonal antibody prepared according to the knowledge and skillsof the field. During the preparation, a test subject was injected withLAG-3 antigen, and then hybridoma expressing antibody which possessesdesired sequence or functional characteristics was separated. In apreferred embodiment of the present invention, the murine LAG-3 antibodyor antigen binding fragment thereof, further comprises light chainconstant region of murine κ, λ chain or a variant thereof, or furthercomprises heavy chain constant region of murine IgG1, IgG2, IgG3, or avariant thereof.

The term “chimeric antibody”, is an antibody which is formed by fusingthe variable region of a murine antibody with the constant region of ahuman antibody, the chimeric antibody can alleviate the murineantibody-induced immune response. To establish chimeric antibody,hybridoma secreting specific murine monoclonal antibody is firstestablished, a variable region gene is then cloned from mouse hybridomacells, then a constant region gene of a human antibody is cloned asdesired, the mouse variable region gene is ligated with human constantregion gene to form a chimeric gene which can be inserted into a humanvector, and finally the chimeric antibody molecule is expressed in theeukaryotic or prokaryotic system. In a preferred embodiment of thepresent invention, the light chain of LAG-3 chimeric antibody furthercomprises the light chain constant regions of human κ, λ chain or avariant thereof The heavy chain of LAG-3 chimeric antibody furthercomprises the heavy chain constant regions of human IgG1, IgG2, IgG3,IgG4 or a variant thereof, preferably comprises heavy chain constantregion of human IgG1, IgG2, IgG3 or IgG4, or preferably comprises heavychain constant region of human IgG1, IgG2 or IgG4, or a variant thereofwith amino acid mutations (e.g., YTE mutations).

The term “humanized antibody”, also known as CDR-grafted antibody,refers to an antibody generated by grafting murine CDR sequences into avariable region framework of a human antibody, namely, an antibodyproduced among different types of human germline antibody frameworksequences. Humanized antibody overcomes the heterogenous responseinduced by the chimeric antibody which carries a large amount of murineprotein components. Such framework sequences can be obtained from publicDNA database covering germline antibody gene sequences or from publishedreferences. For example, germline DNA sequences of human heavy and lightchain variable region genes can be found in “VBase” human germlinesequence database (available on web www.mrccpe.com.ac.uk/vbase), as wellas can be found in Kabat, E A, et al, 1991 Sequences of Proteins ofImmunological Interest, 5th Ed. To avoid the decrease in the activityduring immunogenicity reduction, the variable region frame sequence ofthe human antibody is subjected to a minimum back mutation to maintainthe activity. The humanized antibody of the present invention alsocomprises a humanized antibody which is further subjected to CDRaffinity maturation by phage display. In a preferred embodiment of thepresent invention, the murine CDR sequences of the humanized LAG-3antibody are selected from SEQ ID NOs: 9-20; The variable region frameof human antibody is designed to be selected, wherein the heavy chain FRsequence of the heavy chain variable region of the antibody is derivedfrom the combination sequence of human germline heavy chains IGKV1-39*01and hjk4.1; wherein the light chain FR sequence of the light chainvariable region of the antibody is derived from the combination sequenceof human germline heavy chains IGHV3-23*04 and hjh6.1. In order to avoidthe decrease of the activity caused by the decrease of immunogenicity,the variable region of the human antibody described herein can besubjected to minimal back-mutations to maintain the activity ofantibody.

The grafting of CDRs may result in a decrease in the affinity of theLAG-3 antibody or antigen-binding fragment thereof to the antigen due tothe change of framework residues in contact with the antigen. Suchinteractions may be the result of highly somatic mutations. Thus, it maystill be necessary to implant such donor framework amino acids to theframework of humanized antibodies. The amino acid residues involved inantigen binding from nonhuman LAG-3 antibody or antigen-binding fragmentthereof can be identified by examining the variable region sequence andstructure of murine monoclonal antibody. Each of the residues in the CDRdonor framework that is different from the germline may be considered tobe relevant. If it is not possible to determine the most closely relatedspecies, the sequence can be compared to a consensus sequence of asubtype consensus sequence or a murine sequence with a high similaritypercentage. Rare frame residues are thought to be the result of a highlysomatic cell mutation, which plays an important role in binding.

The term “antigen-binding fragment” of an antibody (or for short,“antibody fragment”), refers to one or more fragments of an antibodythat retain the ability to specifically bind to an antigen (e. g., aLAG-3 protein). It has been shown that the antigen-binding function ofan antibody can be performed by a full length antibody fragment.Examples of binding fragments encompassed within the term “antigenbinding fragment” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge on the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody; (v) a singledomain or a dAb fragment (Ward et al., (1989) Nature10 341:544-546),which consists of a VH domain; and (vi) a separate complementaritydetermining region (CDR) or (vii) optionally a combination of two ormore separate CDRs linked by a synthetic linker. Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen binding fragment” of an antibody. These antibody fragmentsare obtained using conventional techniques known to those with skill inthe art, and the fragments are screened for utility in the same manneras for intact antibodies. The antigen-binding moiety can be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact immunoglobulins. The antibodies may be antibodies of differentisoforms, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype),IgA1, IgA2, IgD, IgE or IgM antibodies.

The term “single chain antibody”, “single chain Fv” or “scFv” isintended to refer to a molecule comprising an antibody heavy chainvariable domain (or region; VH) and an antibody light chain variabledomain (or region; VL) connected by a linker. Such scFv molecules canhave the general structures: NH₂-VL-linker-VH-COOH orNH₂-VH-linker-VL-COOH. A suitable linker in the prior art is composed ofa repetitive GGGGS amino acid sequence or a variant thereof, for examplea variant with 1-4 repeat (Holliger et al. (1993), Proc. Natl. Acad.Sci. USA90: 6444-6448). Other linkers that may be used in the presentinvention are described by Alfthan et al., Protein Eng. 8:725-731, Choiet al (2001), Eur. J. Immuno 1.31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 andRoovers et al (2001), Cancer Immunol.

The term “CDR” refers to one of the six hypervariable regions within thevariable domains of an antibody that mainly contribute to antigenbinding. One of the most commonly used definitions for the six CDRs wasprovided by Kabat E. A. et al, (1991) Sequences of proteins ofimmunological interest. NIH Publication 91-3242). As used herein,Kabat's definition of CDRs only applies for CDR1, CDR2 and CDR3 of thelight chain variable domain (LCDR1, LCDR 2, LCDR 3, or L1, L2, L3), aswell as for CDR2 and CDR3 of the heavy chain variable domain (HCDR2,HCDR3, or H2, H3).

The term “antibody framework”, as used herein refers to the part of thevariable domain, either VL or VH, which serves as a scaffold for theantigen binding loops (CDRs) of this variable domain. In essence it isthe variable domain without the CDRs.

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., particular sites on LAG-3 molecule) to which animmunoglobulin or antibody specifically binds. An epitope typicallyincludes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15consecutive or non-consecutive amino acids in a unique spatialconformation. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G E. Morris, Ed. (1996).

The terms “specific binding,” “selective binding,” “selectively binds,”and “specifically binds,” refer to binding of an antibody to an epitopeon a predetermined antigen. Typically, the antibody binds with anaffinity (1(D) of approximately less than 10⁻⁷ M, such as approximatelyless than 10⁻⁸ M, 10⁻⁹M or 10⁻¹⁰ M or even lower.

The term “competitive binding” refers to an antibody that recognizes thesame epitope (also referred to as an antigenic determinant) or a portionof the same epitope on the extracellular region of human LAG-3 and bindsto the antigen as the monoclonal antibody of the present invention. Anantibody that binds to the same epitope as the monoclonal antibody ofthe present invention refers to an antibody that recognizes and binds tothe amino acid sequence of human LAG-3 recognized by the monoclonalantibody of the present invention.

The term “KD” of “Kd” refers to the dissociation equilibrium constant ofa particular antibody-antigen interaction. Typically, the antibodies ofthe invention bind to LAG-3 with a dissociation equilibrium constant(KD) of less than approximately 10⁻⁷ M, such as less than approximately10⁻⁸M, 10⁻⁹M or 10⁻¹⁰ M or even lower, for example, as determined usingsurface plasmon resonance (SPR) technology in a BIACORE instrument.

The term “nucleic acid molecule” as used herein refers to DNA moleculesand RNA molecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. A nucleic acidis “effectively linked” when it is placed into a functional relationshipwith another nucleic acid sequence. For instance, a promoter or enhanceris effectively linked to a coding sequence if it affects thetranscription of the sequence.

The term “vector,” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. In oneembodiment, vector is a “plasmid,” which refers to a circular doublestranded DNA loop into which additional DNA segments may be ligated. Inanother embodiment, vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. In present invention, thevectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors), or can be integratedinto the genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome (e.g., non-episomalmammalian vectors).

Methods for producing and purifying antibodies and antigen-bindingfragments are well known in the art and can be found, for example, inAntibody Experimental Technology Guide of Cold Spring Harbor, Chapters5-8 and 15. For example, mice can be immunized with human LAG-3, orfragments thereof, and the resulting antibodies can then be re-natured,purified and sequenced by using conventional methods well known in theart. Antigen-binding fragments can also be prepared by conventionalmethods. The antibody or the antigen-binding fragment of the presentinvention is genetically engineered to introduce one or more humanframework regions (FRs) to a non-human derived CDR. Human FR germlinesequences can be obtained from ImMunoGeneTics (IMGT) via their websitehttp://imgt.cines.fr, or from The Immunoglobulin FactsBook,2001ISBN012441351.

The term “host cell” refers to a cell into which an expression vectorhas been introduced. Host cells can include bacterial, microbial, plantor animal cells. Bacteria, which are susceptible to be transformed,include members of the enterobacteriaceae, such as strains ofEscherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis;Pneumococcus; Streptococcus, and Haemophilus influenzae. Suitablemicroorganisms include Saccharomyces cerevisiae and Pichia pastoris.Suitable animal host cell lines include CHO (Chinese Hamster Ovarylines) and NSO cells.

The engineered antibody or antigen-binding fragment of the presentinvention may be prepared and purified using conventional methods. Forexample, cDNA sequences encoding a heavy chain and a light chain may becloned and recombined into a GS expression vector. The recombinantimmunoglobulin expression vector may then stably transfect CHO cells. Asa more recommended method well known in the art, mammalian expressionsystem will result in glycosylation, typically at the highly conservedN-terminus in the FC region. Stable clones are obtained throughexpression of an antibody specifically binding to human LAG-3. Positiveclones may be expanded in a serum-free culture medium for antibodyproduction in bioreactors. Culture medium, into which an antibody hasbeen secreted, may be purified by conventional techniques. For example,the medium may be conveniently applied to a Protein A or G Sepharose FFcolumn that has been equilibrated with a compatible buffer. The columnis washed to remove nonspecific binding components. The bound antibodyis eluted by PH gradient and antibody fragments are detected bySDS-PAGE, and then pooled. The antibody may be filtered and concentratedusing common techniques. Soluble mixture and aggregate may beeffectively removed by common techniques, including size exclusion orion exchange. The obtained product may be immediately frozen, forexample at −70° C., or may be lyophilized.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contacting an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition with the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contacting areagent with the cell, as well as contacting a reagent with a fluid,where the fluid is in contact with the cell. “Administration” and“treatment” also means in vitro and ex vivo treatments, e.g., of a cell,by a reagent, diagnostic, binding compound, or by another cell.“Treatment,” as it applies to a human, veterinary, or a subject to bestudied, refers to therapeutic treatment, prophylactic or preventativemeasures, to research and diagnostic applications.

“Treat” means to administer a therapeutic agent, such as a compositioncomprising any of the binding compounds of the present invention,internally or externally to a patient having one or more diseasesymptoms for which the agent has known therapeutic activity. Typically,the agent is administered in an amount effective to alleviate one ormore disease symptoms in the treated patient or population, whether byinducing the regression of or inhibiting the progression of suchsymptom(s) to any clinically measurable degree. The amount of atherapeutic agent that is effective to alleviate any particular diseasesymptom (also referred to “therapeutically effective amount”) may varyaccording to factors such as the disease state, age, and weight of thepatient, and the ability of the drug to elicit a desired response in thepatient. Whether a disease symptom has been alleviated can be assessedby any clinical measurement typically used by physicians or otherskilled healthcare providers to assess the severity or progressionstatus of that symptom. While an embodiment of the present invention(e.g., a treatment method or article of manufacture) may not beeffective in alleviating the disease symptom(s) of interest in everypatient, it should alleviate the target disease symptom(s) of interestin a statistically significant number of patients as determined by anystatistical test known in the art such as the Student's t-test, thechi-square test, the U-test according to Mann and Whitney, theKruskal-Wallis test (H-test), Jonckheere-Terpstra-test and theWilcoxon-test.

“Conservative modifications” or “conservative replacement orsubstitution” refers to substitutions of amino acids in a protein withother amino acids having similar characteristics (e.g. charge,side-chain size, hydrophobicity/hydrophilicity, backbone conformationand rigidity, etc.), such that the changes can frequently be madewithout altering the biological activity of the protein. Those of skillin this art recognize that, in general, single amino acid substitutionsin non-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. (1987) Molecular Biologyof the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4.sup.th Ed.)). Inaddition, substitutions with structurally or functionally similar aminoacids are less likely to disrupt biological activity.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of a medical condition. Effective amount alsomeans an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular patient or veterinary subject may varydepending on factors such as the condition being treated, the generalhealth of the patient, the route and dose of administration and theseverity of side effects. An effective amount can be the maximal dose ordosing protocol that avoids significant side effects or toxic effects.

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context. “Endogenous” refers tosubstances that are produced within a cell, organism, or human body,depending on the context.

“Homology” refers to sequence similarity between two polynucleotidesequences or between two polypeptides. When a position in both of thetwo sequences to be compared is occupied by the same base or amino acidmonomer subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then the molecules are homologous at that position.The percent of homology between two sequences is a function of thenumber of matching or homologous positions shared by the two sequencesdivided by the number of positions compared multiplying by 100. Forexample, if 6 of 10 positions in two sequences are matched or homologouswhen the sequences are optimally aligned, then the two sequences are 60%homologous. If 95 of 100 positions in two sequences are matched orhomologous when the sequences are optimally aligned, then the twosequences are 95% homologous. Generally, the comparison is made when twosequences are aligned to give maximum percent homology.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without consideringthe number of transfers. It is also understood that all progeny may notbe precisely identity in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific moiety ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of, orbeyond the region of interest needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to the corresponding strands of the template tobe amplified. The 5′ terminal nucleotides of the two primers can beidentical to the ends of the material to be amplified. PCR can be usedto amplify specific RNA sequences, specific DNA sequences from totalgenomic DNA, and cDNA transcribed from total cellular RNA, bacteriophageor plasmid sequences, etc. See generally Mullis et al. (1987) ColdSpring Harbor Symp. Ouant. Biol. 51:263; Erlich, ed., (1989) PCRTECHNOLOGY (Stockton Press, N.Y.). As used herein, PCR is considered asone, but not the only, example of a nucleic acid polymerase reactionmethod for amplifying a nucleic acid test sample, comprising the use ofa known nucleic acid as a primer and a nucleic acid polymerase toamplify or generate a specific moiety of the nucleic acid.

“Optional” or “optionally” means that the event or situation thatfollows may but does not necessarily occur, and the description includesthe instances in which the event or circumstance does or does not occur.For example, “optionally comprises 1-3 antibody heavy chain variableregions” means the antibody heavy chain variable region with specificsequence can be, but not necessarily be present.

“Pharmaceutical composition” refers to a mixture containing one or morecompounds according to the present invention or aphysiologically/pharmaceutically acceptable salt or prodrug thereof andother chemical components, such as physiologically/pharmaceuticallyacceptable carriers and excipients. The pharmaceutical composition aimsat promoting the administration to an organism, facilitating theabsorption of the active ingredient and thereby exerting a biologicaleffect.

EXAMPLE AND TEST

Hereinafter, the present invention is further described with referenceto the examples. However, the scope of the present invention is notlimited thereto. In the examples of the present invention, wherespecific conditions are not described, the experiments are generallyconducted under conventional conditions as described in AntibodyTechnology Laboratory Manual and Molecular Cloning Manual of Cold SpringHarbor, or under conditions proposed by the material or productmanufacturers. Where the source of the reagents is not specificallygiven, the reagents are commercially available conventional reagents.

Example 1 Preparation of LAG-3 Antigen and Antibody 1. Protein Designand Expression

UniProt Lymphocyte activation gene 3 protein (human LAG-3, Uniprot:P18627) was used as the template of the LAG-3, and the amino acidsequences of the antigen and the protein used for detection weredesigned, optionally different labels were fused to the LAG-3 proteinand then cloned into pHr vector (produced in-house) or pTT5 vector(Biovector, Cat#: 102762) or pTargeT vector (Promega, A1410). Theantigen protein and the detection protein of the present invention weretransiently expressed in 293 cells or stably expressed in CHO-S,purified and obtained.

The following LAG-3 antigens are referred to human LAG-3 if notspecifically described.

LAG-3 Extracellular domain with a Flag tag: LAG-3-Flag, for immunizationof mice.

SEQ ID NO: 1 MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSP GDYKDDDDK

NOTE: Underlined sequence represents a signal peptide, and italic partrefers to the Flag-tag sequence.

The full length sequence of LAG-3: Used to construct LAG-3overexpressing cell line, for immunization of mice and detection

SEQ ID NO: 2 MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGF HLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL

NOTE: Signal peptide+extracellular domain+transmembraneregion+intracellular domain

A fusion protein of LAG-3 extracellular domain and hIgG1 Fc: LAG-3-Fc,for detection

SEQ ID NO: 3 MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSS PGDDDDKGSGSGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG

NOTE: Underlined sequence represents a signal peptide, double underlinedsequence represents a linker, and the italic part represents Fc.

A fusion protein of LAG-3 extracellular domain and mIgG2a Fc:

LAG-3-mFc, for detection

SEQ ID NO: 4 MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSS PGDDDDKGSGSGEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFS RTPGK

NOTE: Underlined sequence represents a signal peptide, double underlinedsequence represents a linker, and the italic part represents Fc.

2. Purification of LAG-3-Related Recombinant Protein, as Well asHybridoma Antibody, and Recombinant Antibody 1. Purification Steps forLAG-3-Flag Recombinant Protein with a Flag Tag

The sample was centrifuged at high speed to remove impurities andconcentrated to an appropriate volume. After that, the flag affinitycolumn was equilibrated with 0.5×PBS and washed with 2-5 column volumes.The supernatant samples were loaded on the column after removing theimpurity. Washing the column with 0.5×PBS until the A280 reading wasreduced to baseline. Then, the column was washed with PBS, and theimpurity protein was washed off and then collected. The target proteinwas eluted with 100 mM glycine, pH 3.0 and collected for furtheractivation and purification in vitro.

2. Purification Steps for Hybridoma Antibody, Recombinant Antibody andFc Fusion Protein

The cell-expressing supernatant was centrifuged at high speed to removeimpurities, hybridoma expressing supernatant was purified by Protein Gcolumn, recombinant antibody and Fc fusion protein were purified byProtein A column. Washing the column with 0.5×PBS until the A280 readingwas reduced to baseline. After that, the target protein was eluted with100 mM acetic acid (pH 3.0) and neutralized with 1 M Tris-HCl, pH 8.0.The eluted sample was properly concentrated and further purified usinggel chromatography Superdex200 (GE), which was equilibrated with PBS,the mismatch peak was excluded and the correct sample was aliquoted foruse.

Example 2 Preparation of Anti-Human LAG-3 Monoclonal Antibody 1.Immunization

The anti-human LAG-3 monoclonal antibody was produced by immunizingmice. Experimental SJL white mice, female, 6-week old (Beijing WeitongLihua Experimental Animal Technology Co., Ltd., animal productionlicense number: SCXK (Beijing) 2012-0001). Feeding environment: SPFlevel. After the mice were purchased, the animals were kept in thelaboratory for 1 week, with 12/12-hour light/dark cycle, at temperatureof 20-25° C., and with a humidity of 40-60%. The mice that had beenadapted to the environment were immunized according to the followingschemes. Immune antigen was extracellular domain of LAG-3 with Flag tag(SEQ ID NO: 1).

Scheme A: Mice were cross-immunized with TiterMax® Gold Adjuvant (sigmaLot Num: T2684) and Thermo Imject® Alum (Thremo Lot Num: 77161). Theratio of antigen to adjuvant (TiterMax® Gold Adjuvant) was 1:1, and theratio of antigen to adjuvant (Thermo Imject® Alum) was 3:1, with a doseof 50 μg/mouse (first immunization) and 25 μg/mouse (boosterimmunization). After the antigen was emulsified, the mice wereinoculated on day 0, 7, 14, 21, 28, 35 and 42. On day 0, the mice were,on several locations, subcutaneously (s.c.) injected with emulsifiedantigen, 50 μg/mouse. On day 7, the mice were intraperitoneally (i.p.)injected with 25 μg/mouse. On days 14, 28, 35 and 42, either back orintraperitoneal injection of antigen was selected according to the lumpson the back and the swelling conditions in abdomen. Blood samples werecollected on days 21, 35, 49, and antibody titers in mouse serum weredetermined by ELISA. After 7 immunizations, mice with higher antibodytiter and the titer tending to platform in their serum were selected forsplenocyte fusion, a booster immunization was performed by i.p.injection of antigen solution formulated with saline, 50 μg/mouse, 3days prior to splenocyte fusion.

Scheme B: Mice were immunized with QuickAntibody-Mouse5W (KX0210041).The ratio of antigen to adjuvant was 1:1, 25 μg/mouse (firstimmunization/booster immunization). The antigen and adjuvant wererapidly mixed and used for inoculation. Mice were inoculated on days 0,21 and 35. On day 0, the mouse was injected with antigens via posteriorcalf muscles (i.m.), 25 μg/mouse, On days 21 and 35, the same way ofinjection was repeated, 25 μg/mouse, (whether the third immunization wasperformed or not is dependent on the antibody titer). Blood samples werecollected on days 28 and 42. The antibody titer in mouse serum wasdetermined by ELISA. Mice with higher antibody titer and the titertending to platform in their serum were selected for splenocyte fusion,a booster immunization was performed by i.p. injection of antigensolution formulated with saline, 50 μg/mouse, 3 days prior to splenocytefusion.

2. Splenocyte Fusion

Hybridoma cells were obtained by fusing splenic lymphocyte with myelomaSp2/0 cells (ATCC® CRL-8287™) by using an optimized PEG-mediated fusionprocedure. The hybridoma cells obtained were resuspended in a completemedium (DMEM medium containing 20% FBS, 1×HAT and 1×OPI) at a density of0.5-1×10⁶/ml, and incubated in 96-well cell culture plates, 100 μl/well.After incubation at 37° C., 5% CO₂, for 3-4 days, 100 μl/well of the HATcomplete medium was supplemented and the culture was continued for 3-4days to form a needle-like clone. The supernatant was removed and 200μl/well of HT complete medium (RPMI-1640 medium containing 20% FBS,1×HAT, 1×OPI) was added, cultured at 5% CO₂, 37° C. for three days andthen detected by ELISA assay.

3. Screening for Hybridoma Cells

Hybridoma culture supernatant was detected by binding ELISA (see TestExample 1) according to the growth density of hybridoma cells. Andcell-blocking experiments were performed with positive wells of ELISA(see Test Example 3). Cells which were positive both for binding andblocking experiments were expanded and frozen stored in timely, and thecells were subcloned twice to three times until single cell clone wasobtained.

After each subcloning procedure, the cells were subjected to LAG-3binding

ELISA and cell blocking assay (see Test Example 1 and Test Example 3).The hybridoma clones were obtained by the above screening experiments,and the antibody was further prepared by serum-free cell culture method,and then the antibody was purified according to purification example foruse in the test example.

4. Sequencing of the Positive Hybridoma Clone

The process of cloning sequence from the positive hybridoma was asfollows: Collecting the hybridoma cells at logarithmic growth phase, andextracting RNA with Trizol (Invitrogen Cat, No. 15596-018) according tothe kit instructions, and then performing reverse transcription with thePrimeScript™ Reverse Transcriptase kit (Takara, Cat No. 2680A). ThecDNAs obtained by reverse transcription were amplified by PCR using themouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequencing wasperformed in a sequencing company. The heavy chain and light chain aminoacid sequences corresponding to DNA sequences of hybridoma clone mAb229and mAb303 are shown in SEQ ID NOs: 5, 6 and SEQ ID NOs: 7, 8,respectively.

mAb229-VH SEQ ID NO: 5 QIQLVQSGPELKKPGETVKISCKASGYTFTTSGMSWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDNYDARDVYYYAMDYWGQGTSVTVSS mAb229-VL SEQ ID NO: 6DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWITPW TFGGGTKLEIK mAb303-VHSEQ ID NO: 7 EVQLQQSGPVLVKPGASVKMSCKASGYTLTDYYMNWVKQSHGKSLEWIGVINPYNGDTAYNQKFKGKATLTVDKSSNTAYMEINSLTSEDSAVYYCTRDDGYYDYYFDVWGTGTTVTVSS mAb303-VL SEQ ID NO: 8DIQMTQSPSSLSASLGERVILTCRASQDIGSRLNWLQQGPDGTFKRLIYATSTLDSGVPKRFSGSRSGSDFSLTISSLESEDFVDYYCLQLASSPP TFGGGTKLEIK

TABLE 1  CDR region sequences of heavy chain and light chain Heavy chainLight chain mAb229 HCDR1 TSGMS LCDR1 RASENIYSNLA SEQ ID NO: 9SEQ ID NO: 15 HCDR2 WINTYSGVPTYADDFKG LCDR2 AATNLAD SEQ ID NO: 10SEQ ID NO: 16 HCDR3 DNYDARDVYYYAMDY LCDR3 QHFWITPWT SEQ ID NO: 11SEQ ID NO: 17 mAb303 HCDR1 DYYMN LCDR1 RASQDIGSRLN SEQ ID NO: 12SEQ ID NO: 18 HCDR2 VINPYNGDTAYNQKFKG LCDR2 ATSTLDS SEQ ID NO: 13SEQ ID NO: 19 HCDR3 DDGYYDYYFDV LCDR3 LQLASSPPT SEQ ID NO: 14SEQ ID NO: 20

The obtained positive clones were subjected to an ELISA assay of bindingto human LAG-3 (Test Example 1, the results of EC50 value for theprotein binding activity are shown in Table 2), ELISA assay of bindingto human LAG-3 overexpressing CHO-s cells (Test Example 2, the resultsof EC50 values for the cell binding activity are shown in Table 2), andan assay for blocking the binding of LAG-3 antigen to Daudi cells (TestExample 3, the results of EC50 value for blocking activity are shown inTable 2), and assay for its affinity with human LAG-3 protein (see TestExample 4, results are shown in Table 3).

TABLE 2 In vitro Activity of Murine LAG-3 Antibody Candidate Proteinbinding Cell binding Blocking activity antibody activity EC50(nM)activity EC50(nM) IC50 (nM) mAb229 0.129 0.191 1.327 mAb303 0.172 0.2790.596

TABLE 3 Affinity of Murine LAG-3 antibody Stationary phase Mobile phaseAffinity(M) mAb229 LAG-3-Flag 4.26E−10 mAb303 4.70E−10

The results shown in table 2 demonstrate that both the LAG-3 antibodymAb229 and mAb303 showed excellent binding activity to human LAG-3protein and the two also showed excellent binding activity to CHO-Scells overexpressing full-length of human LAG-3 protein. Both LAG-3antibody mAb229 and mAb303 significantly blocked the binding of humanLAG-3 antigen with Daudi cells.

The results shown in table 3 demonstrate that the LAG-3 antibody mAb229and mAb303 of the present invention showed a stronger binding activityand affinity to human LAG-3 protein.

Example 3 Humanization of Murine Anti-Human LAG-3 Hybridoma MonoclonalAntibody mAb229

Through comparison in the IMGT human antibody heavy and light chainvariable region gene database and MOE software, the heavy and lightchain variable region germline genes with high homology to mAb229 wereselected as templates, the CDRs derived from murine antibodies weregrafted into the corresponding human source template to form a variableregion sequence with the order in FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.Wherein, amino acid residues were identified and annotated according toKabat Numbering System.

1. Selection of a Framework for Humanizing Hybridoma Clone mAb229

The light chain template for humanizing murine antibody mAb229 isIGKV1-39*01 and hjk4.1, the heavy chain template for humanization isIGHV7-4-1*01 and hjh6.1, the sequences of humanized variable region areas follows:

Hu229VH-CDR graft SEQ ID NO: 21 QVQLVQSGSELKKPGASVKVSCKASGYTFT TSGMSWVRQAPGQGLEWM G WINTYSGVPTYADDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYC ARDNYDARDVYYYAMDY WGQGTTVTVSS Hu229VL-CDR graft SEQ ID NO: 22DIQMTQSPSSLSASVGDRVTITC RASENIYSNLA WYQQKPGKAPKLLI Y AATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHFWITPW T FGGGTKVEIK

NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italic sequencerepresents FR sequence, and underlined sequence represents CDR sequence.

2. Template Selection and Back-Mutation Design for Hybridoma ClonemAb229, see Table 4 below

Hu229_VL Hu229_VH Hu229_VL.1 Grafted Hu229_VH.1 Grafted Hu229_VL.1AI48V, F71Y Hu229_VH.1A E46K Hu229_VL.1B D70Q, F71Y, Hu229_VH.1B E46K,R38K, I48V V93T Hu229_VL.1C D70Q, F71Y, Hu229_VH.1C E46K, R38K, I48V,A43S V93T, Y95F NOTE: For example, I48V denotes a back mutation from Ito V at position 48 according to Kabat numbering system. Graftedindicates that the murine antibody CDR was implanted into human germlineFR sequences.

TABLE 5 Sequence combinations for humanizing murine antibody mAb229Hu229_VL.1 Hu229_VL.1A Hu229_VL.1B Hu229_VL.1C Hu229_VH.1 Hu229-004 LF229-005 Hu229-006 Hu229-007 Hu229_VH.1A Hu229-008 Hu229-009 Hu229-010Hu229-011 Hu229_VH.1B Hu229-012 Hu229-013 Hu229-014 Hu229-015Hu229_VH.1C Hu229-016 Hu229-017 Hu229-018 Hu229-019 NOTE: This tableshows various sequence combinations of different mutations. For example,Hu229-005 indicates that two mutations (light chain HumAb229_VL.1A andheavy chain HumAb229_VH.1) are present in the humanized murine antibodyHu229-005, and so on.

Sequences of humanized antibody mAb229 are as follows:

Hu229VH.1 (identical to Hu229VH-CDR graft) SEQ ID NO: 21QVQLVQSGSELKKPGASVKVSCKASGYTFTTSGMSWVRQAPGQGLEWMGWINTYSGVPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDNYDARDVYYYAMDYWGQGTTVTVSS Hu229VH.1A SEQ ID NO: 23QVQLVQSGSELKKPGASVKVSCKASGYTFTTSGMSWVRQAPGQGLKWMGWINTYSGVPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDNYDARDVYYYAMDYWGQGTTVTVSS Hu229VH.1B SEQ ID NO: 24QVQLVQSGSELKKPGASVKVSCKASGYTFTTSGMSWVKQAPGQGLKWMGWINTYSGVPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTATYYCARDNYDARDVYYYAMDYWGQGTTVTVSS Hu229VH.1C SEQ ID NO: 25QVQLVQSGSELKKPGASVKVSCKASGYTFTTSGMSWVKQAPGQGLKWMGWINTYSGVPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTATYFCARDNYDARDVYYYAMDYWGQGTTVTVSS Hu229VL.1 (identical to Hu229VL-CDR graft)SEQ ID NO: 22 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWITPWTFGG GTKVEIK Hu229VL.1ASEQ ID NO: 26 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLVYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWITPWTFGG GTKVEIK Hu229VL.1BSEQ ID NO: 27 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLVYAATNLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHFWITPWTFGG GTKVEIK Hu229VL.1CSEQ ID NO: 28 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHFWITPWTFGG GTKVEIK

Example 4 Humanization of Murine Anti-Human LAG-3 Hybridoma MonoclonalAntibody mAb303

Through comparison in the IMGT human antibody heavy and light chainvariable region gene database and MOE software, the heavy and lightchain variable region germline genes with high homology to mAb303 wereselected as templates, the CDRs derived from murine antibodies weregrafted into the corresponding human source template to form a variableregion sequence with the order in FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.Wherein, amino acid residues were identified and annotated according tothe Kabat Numbering System.

1. Selection of a Framework for Humanizing Hybridoma Clone mAb303

The light chain template for humanizing murine antibody mAb303 isIGKV1-39*01 and hjk4.1, the heavy chain template for humanization isIGHV1-3*01 and hjh6.1, the sequences of humanized variable region are asfollows:

Hu303VH-CDR graft SEQ ID NO: 29 QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYMNWVRQAPGQRLEWM G VINPYNGDTAYNQKFKG RVTITRDTSASTAYMELSSLRSEDTAVYYC ARDDGYYDYYFDV WGQGTTVTVSS Hu303VL-CDR graft SEQ ID NO: 30DIQMTQSPSSLSASVGDRVTITC RASQDIGSRLN WYQQKPGKAPKLLI Y ATSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQLASSPP T FGGGTKVEIK

NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italic sequencerepresents FR sequence, and the underlined sequence represents CDRsequence.

2. Template Selection and Back-Mutation Design of Hybridoma ClonemAb303, See Table 6 Below

Hu303_VL Hu303_VH Hu303_VL.1 Grafted Hu303_VH.1 Grafted Hu303_VL.1AL46R, G66R Hu303_VH.1A R72V, T74K, A97T Hu303_VL.1B L46R, G66R,Hu303_VH.1B R72V, T74K, S60K A97T, F29L Hu303_VL.1C L46R, G66R,Hu303_VH.1C R72V, T74K, S60K, P44F, F29L, A97T, Y36L M48I, V68A, I70LHu303_VL.1D L46R, G66R, S60K, P44F, Y36L, K42G, I21L, T85D NOTE: Forexample, L46R denotes a back mutation from L to R at position 46according to Kabat numbering system. Grafted indicates that the murineantibody CDR was implanted into human germline FR sequences.

Sequence combinations of different mutations are as follows:

TABLE 7 Sequence combinations for humanization of murine antibody mAb303Hu303_VL.1 Hu303_VL.1A Hu303_VL.1B Hu303_VL.1C Hu303_VL.1D Hu303_VH.1Hu303-004 Hu303-005 Hu303-006 Hu303-007 Hu303-008 Hu303_VH.1A Hu303-009Hu303-010 Hu303-011 Hu303-012 Hu303-013 Hu303_VH.1B Hu303-014 Hu303-015Hu303-016 Hu303-017 Hu303-018 Hu303_VH.1C Hu303-019 Hu303-020 Hu303-021Hu303-022 Hu303-023 NOTE: This table shows various sequence combinationsof different mutations. For example, Hu303-005 indicates that twomutations (light chain HumAb303_VL.1A and heavy chain HumAb303_VH.1) arepresent on the humanized murine antibody Hu303-005, and so on.

Sequences of humanized antibody mAb303 are as follows:

Hu303_VH.1 (identical to Hu303VH-CDR graf) SEQ ID NO: 29QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQRLEWMGVINPYNGDTAYNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR DDGYYDYYFDVWGQGTTVTVSSHu303_VH.1A SEQ ID NO: 31QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQRLEWMGVINPYNGDTAYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCTR DDGYYDYYFDVWGQGTTVTVSSHu303_VH.1B SEQ ID NO: 32QVQLVQSGAEVKKPGASVKVSCKASGYTLTDYYMNWVRQAPGQRLEWMGVINPYNGDTAYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCTR DDGYYDYYFDVWGQGTTVTVSSHu303_VH.1C SEQ ID NO: 33QVQLVQSGAEVKKPGASVKVSCKASGYTLTDYYMNWVRQAPGQRLEWIGVINPYNGDTAYNQKFKGRATLTVDKSASTAYMELSSLRSEDTAVYYCTR DDGYYDYYFDVWGQGTTVTVSSHu303_VL.1 (identical to Hu303VL-CDR graft) SEQ ID NO: 30DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWYQQKPGKAPKLLIYATSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQLASSPPTF GGGTKVEIK Hu303_VL.1ASEQ ID NO: 34 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWYQQKPGKAPKRLIYATSTLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQLASSPPTF GGGTKVEIK Hu303_VL.1BSEQ ID NO: 35 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWYQQKPGKAPKRLIYATSTLDSGVPKRFSGSRSGTDFTLTISSLQPEDFATYYCLQLASSPPTF GGGTKVEIK Hu303_VL.1CSEQ ID NO: 36 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAFKRLIYATSTLDSGVPKRFSGSRSGTDFTLTISSLQPEDFATYYCLQLASSPPTF GGGTKVEIK Hu303_VL.1DSEQ ID NO: 37 DIQMTQSPSSLSASVGDRVTLTCRASQDIGSRLNVVLQQKPGGAFKRLIYATSTLDSGVPKRFSGSRSGTDFTLTISSLQPEDFADYYCLQLASSPPT FGGGTKVEIK

Example 5 Preparation of Recombination and Humanized Antibody

The antibody was constructed with constant region derived from humanheavy chain IgG4/light chain kappa in combination with each variableregion, and a S228P mutation was made in Fc to increase the stability ofthe IgG4 antibody. The other mutations known in the art can also be usedto increase its performance.

Constant region of heavy chain: SEQ ID NO: 38ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Constant region of light chain:SEQ ID NO: 39 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

1. Molecular Cloning of the Recombinant Antibody

The variable region coding sequences were obtained by sequencing thepositive antibody molecules obtained from hybridoma screening. Theprimers were designed according to the obtained sequence, the sequencinggene was used as template, and various antibody VH/VK gene fragmentswere constructed by PCR, and then reconstituted with the expressionvector pHr (with a signal peptide and hIgG4/hkappa constant region(CH1-FC/CL) fragment) to construct an expression plasmidVH-CH1-FC-pHr/VL-CL-pHr for full-length recombinant antibody.

2. Molecular Cloning of Humanized Antibody

The designed humanized antibody sequence was subjected to codonoptimization and a coding sequence with human codon preference wasgenerated. Primers were designed and various VH/VK gene fragments of theantibodies were constructed by PCR, and reconstituted with theexpression vector pHr (with a signal peptide and hIgG4/hkappa constantregion (CH1-FC/CL) fragment) to construct an expression plasmidVH-CH1-FC-pHr/VL-CL-pHr for full-length humanized antibody.

3. Expression and Purification of Recombination and Humanized Antibody

The plasmids for separate expression of antibody light chain and heavychain were co-transfected into HEK293E cell at a ratio of 1:1.2. Theexpression supernatant was collected after 6 days and impurities wereremoved by high-speed centrifugation and then purified by Protein Acolumn. The column was washed with PBS until the A280 reading wasreduced to baseline. The target protein was eluted with acidic elutionbuffer, pH 3.0-pH 3.5, and neutralized with 1 M Tris-HCl, pH 8.0-9.0.The eluent was properly concentrated and further purified by gelchromatography Superdex200 (GE) which was equilibrated with PBS. Themismatch peak was excluded and the elution peak was collected. Then thecorrect sample was aliquoted and for use.

The performance and benefits of the antibody of the present inventionare verified by biochemical test methods as below.

Test Example 1 ELISA Assay for the Binding of LAG-3 Antibody to HumanLAG-3 Protein

The binding ability of anti-LAG-3 antibody to human LAG-3 protein wasdetected by ELISA assay. LAG-3 fusion protein with Fc or mFc tag wasimmobilized into 96-well microtiter plate by binding to anti-Fc or mFcantibody coated in the microtiter plate, the strength of the signalafter the addition of the antibody was used to determine the bindingactivity of the antibody to LAG-3, the specific experimental method isas follows.

The goat anti-human Fc antibody (Jackson Immuno Research, Cat No.109-005-008) or goat anti-mouse Fc antibody (Sigma, Cat No. M3534-1ML)was diluted to a concentration of 2 μg/ml with PBS buffer at pH 7.4, andadded to a 96-well plate at a volume of 50 μl/well and then, the platewas incubated in the incubator at 37° C. for 2 hours. After discardingthe liquid, the plates were blocked with 200 μl/well of blockingsolution containing 5% skim milk (Guangming skim milk) in PBS, andincubated in the incubator at 37° C. for 2.5 hours or overnight at 4° C.(16-18 hours). After blocking, the blocking solution was discarded andthe plate was washed 5 times with PBST buffer (PH7.4 PBS containing0.05% tweeen-20). LAG-3-Fc fusion protein (SEQ ID NO:3, producedin-house) or LAG-3-mFc fusion protein (SEQ ID NO: 4, produced in-house)was diluted with sample dilution (PH7.4 PBS containing 1% BSA) to 1μg/ml and was added to each well, 50 μl/well. Then the plate wasincubated in the incubator at 37° C. for 1 h or overnight at 4° C. Afterincubation, the reaction solution in the plate was discarded, and theplate was washed with PBST for 6 times, and then was added with 50 μl ofvarious concentrations of the test antibody (hybridoma purified antibodyor humanized antibody) diluted with sample dilution and the plate wasincubated at 37° C. for 1 h. The plates was washed 5 times with PBSTafter incubation, and was added with 100 μl/well of goat anti-mouse(Jackson Immuno Research, Cat No. 115-035-003) or goat anti-humansecondary antibody (Jackson Immuno Research, Cat No. 109-035-003)labeled with HRP, diluted in sample dilution, and the plate wasincubated at 37° C. for 1 h. After washing the plates 6 times with PBST,50 μl/well of TMB chromogenic substrate (KPL, Cat No. 52-00-03) wasadded to each well, and incubated at room temperature for 5-15 min, thereaction was stopped by the addition of 50 μl/well 1M H2504 to eachwell. The OD value at a wavelength of 450 nm was read on NOVOStarmicroplate reader, and then EC50 values of the binding of LAG-3 antibodyto human LAG-3 were calculated. The results are shown in Table 8. Thedata showed that all the humanized antibodies obtained by the screeningmethod in the present invention showed excellent binding activities tohuman LAG-3 protein.

TABLE 8 Determination of EC50 value for Candidate Antibody in BindingAssay Candidate Antibody Binding ELISA EC50(nM) mAb229 0.129 Hu229-0080.506 Hu229-009 0.152 Hu229-010 0.174 Hu229-011 0.201 Hu229-012 0.268Hu229-013 0.106 Hu229-014 0.153 Hu229-015 0.156 Hu229-016 0.154Hu229-017 0.048 Hu229-019 0.068 mAb303 0.172 Hu303-004 0.278 Hu303-0050.309 Hu303-006 0.288 Hu303-007 0.135 Hu303-008 0.140 Hu303-009 0.316Hu303-010 0.137 Hu303-011 0.314 Hu303-012 0.164 Hu303-013 0.166Hu303-014 0.232 Hu303-015 0.172 Hu303-016 0.161 Hu303-017 0.168Hu303-018 0.244 Hu303-019 0.277 Hu303-020 0.140 Hu303-021 0.170Hu303-022 0.145 Hu303-023 0.152

Test Example 2 Binding Assay of LAG-3 Antibody with Human LAG-3Over-Expressing CHO-s Cells

The binding ability of anti-LAG-3 antibody to LAG-3 proteinover-expressing CHO-S cells was detected by binding assay. Thefull-length LAG-3 plasmid (produced in-house, SEQ ID NO: 2) wastransfected into CHO-S cells by electroporation, and the expressionlevel of LAG-3 was detected after two weeks of post-pressure screening.The LAG-3 over-expressing cells were fixed to the bottom of the 96-wellplate, and the strength of the signal after the addition of the antibodywas used to determine the binding activity of the antibody to humanLAG-3 over-expressing CHO-s cells, the specific experimental method isas follows.

100 μl/well of cells were seeded into 96-well plate with a density of4×10⁵/ml and incubated overnight. The supernatant was discarded, and theplate was washed three times with PBS, added with 4% PFA, 100 μl/well,to fix for half an hour at room temperature, and then the plate waswashed three times with PBS. After discarding the liquid, the plate wasblocked with 200 μl/well of blocking solution containing 5% skim milk(Guangming skim milk) diluted in PBS, and incubated at 37° C. for 2.5hours. After blocking, the blocking solution was discarded and the platewas washed 5 times with PBST buffer (PH7.4 PBS containing 0.05%tweeen-20), added with 50 μl/well of test antibody (Hybridoma purifiedantibody or humanized antibody) with different concentrations dilutedwith sample dilution, and then incubated in incubator at 37° C. for 1 h.The plate was washed 5 times with PBST after incubation, added with 100μl/well of goat anti-mouse (Jackson Immuno Research, Cat No.115-035-003) or goat anti-human secondary antibody (Jackson ImmunoResearch, Cat No. 109-035-003) labeled with HRP, diluted in sampledilution, and the plate was incubated at 37° C. for 1 h. After washingthe plates 6 times with PBST, 50 μl of TMB chromogenic substrate (KPL,Cat No. 52-00-03) was added to each well, and incubated at roomtemperature for 5-15 min, the reaction was stopped by the addition of 50μl 1M H₂SO₄to each well. The OD value at a wavelength of 450 nm was readon NOVOStar microplate reader, and then the EC50 values of the bindingof LAG-3 antibody to LAG-3 over-expressing CHOs cell were calculated.

Test Example 3 Assay for the Anti-LAG-3 Antibody in Blocking the Bindingof LAG-3 Antigen to Daudi Cells

Daudi cells (human leukemia cells, purchased from the Chinese Academy ofSciences cell bank) were seeded in 96-well plate with a density of3×10⁵/well. After centrifugation at 1000 rpm, the supernatant wasdiscarded and then the plate was added with 4% PFA to fix for 30 minutesat room temperature. The plate was washed 4 times with PBS afterdiscarding the fixed solution, and the plate was blocked with 200μl/well of blocking solution containing 5% skim milk (Guangming skimmilk) diluted in PBS, and incubated at 37° C. for 2.5 hours. Afterblocking, the blocking solution was discarded and the plate was washed 5times with PBST buffer (PH7.4 PBS containing 0.05% tweeen-20), addedwith 50 μl/well mixture of biotin-labeled LAG-3-Fc fusion protein(produced in-house, SEQ ID NO: 3), diluted with dilution solution (PH7.4PBS containing 1% BSA) at final concentration of 0.4 μg/ml, wherein thebiotin (Biotin labeling kit, Dojindo Chemical, Cat No. LK03) waspre-mixed for an hour, and gradient concentrations of the antibody to betested, then the plate was incubated at 37° C. for 1 h. The reactionsolution was discarded and the plate was washed 5 times with PBST afterincubation, added with 50 μl/well of HRP-labeled Streptavidin (Sigma,Cat No. 52438) which was diluted with sample dilution and the plate wasincubated at 37° C. for 1 h. After washing the plate 5 times with PBST,50 μl/well of TMB chromogenic substrate (KPL, Cat No. 52-00-03) wasadded to each well, and incubated at room temperature for 5-15 min, thereaction was stopped by the addition of 50 μl 1M H₂SO₄ to each well. TheOD value at a wavelength of 450 nm was read on NOVOStar microplatereader, and then the activity of the LAG-3 antibody in blocking thebinding of the antigen to Daudi cells was calculated. The results areshown in Table 9. The data show that all of the humanized antibodiesobtained by the screening method in the present invention significantlyblocked the binding of human LAG-3 antigen to Daudi cells.

TABLE 9 Determination of IC50 value of Candidate Antibody in BlockingAssay Candidate Antibody Binding assay IC50 (nM) mAb229 1.327 Hu229-0090.559 Hu229-010 0.453 Hu229-011 0.566 Hu229-013 0.39 Hu229-014 0.718Hu229-015 0.808 Hu229-016 0.875 Hu229-017 0.239 Hu229-019 0.289 mAb3030.596 Hu303-004 0.502 Hu303-005 0.622 Hu303-006 0.821 Hu303-007 0.343Hu303-008 0.346 Hu303-009 0.417 Hu303-010 0.346 Hu303-011 0.728Hu303-012 0.361 Hu303-013 0.347 Hu303-014 0.467 Hu303-015 0.398Hu303-016 0.395 Hu303-017 0.398 Hu303-018 0.608 Hu303-019 0.471Hu303-020 0.345 Hu303-021 0.456 Hu303-022 0.360 Hu303-023 0.369

Test Example 4 BIAcore Assay for the Affinity of LAG-3 Antibody

1. The mouse anti-capture antibody was covalently linked to the CM5biochip (Cat. # BR-1000-12, GE) according to the method described in themouse anti-capture kit (Cat. #BR-1008-38, GE), so that the test antibodywas captured via affinity. Then, the LAG-3-Flag antigen (producedin-house, SEQ ID NO:1) was flowed through the surface of the biochip,and the reaction signal was detected in real time by using a Biacoreinstrument to obtain the binding and dissociation curves, the value ofaffinity was obtained by fitting, see above table 2. After each cycle ofdissociation is finished in the experiment, the biochip was washed andregenerated with a regeneration solution provided in the mouseanti-capture kit. The results demonstrate that the LAG-3 antibody mAb229and mAb303 showed excellent binding activity and affinity to human LAG-3protein.

2. The human anti-capture antibody was covalently linked to the CM5biochip (Cat. # BR-1000-12, GE) according to the method described in thehuman anti-capture kit (Cat. # BR-1008-39, GE), so that the testantibody was captured via affinity. Then, the LAG-3-Flag antigen(produced in-house, SEQ ID NO:1) was flowed through the surface of thebiochip, and the reaction signal was detected in real time using aBiacore instrument to obtain the binding and dissociation curves, thevalue of affinity was obtained by fitting, see table 10 below. Aftereach cycle of dissociation is finished in the experiment, the biochipwas washed and regenerated with a regeneration solution provided in thehuman anti-capture kit. The results demonstrate that the antibodiesobtained by the screening method in present invention showed excellentbinding activity and affinity to human LAG-3 protein.

TABLE 10 Affinity of anti-LAG-3 Antibody Stationary phase Mobile phaseAffinity(M) mAb229 LAG-3-Flag 1.72E−11 Hu229-009 4.88E−11 Hu229-0103.82E−11 Hu229-013 2.81E−11 Hu229-014 3.74E−11 Hu229-015 4.59E−11Hu229-017 6.71E−11 Hu229-019 7.29E−11 mAb303 7.49E−11 Hu303-004 1.06E−09Hu303-005 7.15E−11 Hu303-006 7.53E−11 Hu303-009 9.43E−10 Hu303-0101.47E−10 Hu303-014 4.91E−10 Hu303-016 7.48E−11

Test Example 5 Activation of PBMC-T Lymphocytes

In order to study the effect of LAG-3 antibody on activating Tlymphocytes, human peripheral blood mononuclear cells (PBMCs) werecollected and purified. The secretion level of IL-2 cytokines wasmeasured after stimulated with super-antigen of Staphylococcus aureusenterotoxin B (SEB) in vitro for 72 h. The experimental process isbriefly described below:

Freshly isolated and purified PBMCs were seeded into 96-well cellculture plate with a cell density of about 1×10⁵/well, and 100 ng/ml SEBsuper-antigen stimulus was added, and gradiently diluted antibodysamples (diluted with medium) or medium as a blank control were added atthe same time. Then, the plate was incubated at 37° C., 5% CO₂ for 72 h,the cell culture supernatant was collected. The level of the secretedIL-2 in the culture supernatant was measured by ELISA (BD, CAT #550611). Detailed procedures are indicated in the manufacturers' manual.The result was shown in FIG. 1. Both humanized LAG-3 antibodiesHu229-013 and Hu303-005 can enhance the levels of cytokine IL-2 secretedby the activated T lymphocytes to different degree, and hasdose-depending effect of drug concentration.

Test Example 6 Inhibition of Subcutaneously Inoculated U-87MG Tumor byLAG-3 Antibody

In this study, the effect of humanized anti LAG-3 antibody on the tumorvolume of U-87 MG tumor bearing mice was measured.

100 μl of human glioma U87 MG cells (3.5×10⁶ cells) were inoculatedsubcutaneously in right ribs of NOD-SCID mice (Purchased from ChangzhouCavion Experimental Animal Co., Ltd.). When the tumor grew to 40 mm³after 10 to 14 days, the mice, excluding ones with too large or toosmall body weight or tumor volume, were randomly divided into threegroups: a control group with Isotype matched hIgG, a group with LAG-3candidate antibodies Hu229-013 and a group with LAG-3 candidateantibodies Hu303-005, according to the tumor volume (Grouping and dosagesee Table 1), each group of 8 mice (DO). The PBMCs stimulated by CD3antibody were injected into the tumor tissues at 5×10⁵ cells/60 μl, andthe injection of test antibody was started via i.p. injection, threetimes a week for total of 6 times.

Mice were measured for tumor volume twice a week, data were recorded.Tumor volume (V) was calculated as:

Tumor volume (TV)=½×a×b ², wherein a and b represent length and width,respectively.

The tumor volume of each group was expressed as mean±standard error(Mean±SEM), and plotted with Graphpad Prism 5 software, analyzed withtwo way ANOVA statistical analysis, and the tumor inhibition rate wascalculate according to the following formula:

Tumor proliferation rate (T/C%)=(T−T ₀ /C−C ₀)×100%

Tumor inhibition rate % TGI=1−T/C %

The result was shown in table 11 and FIG. 2, indicating that 14-day postadministration, LAG-3 antibodies Hu229-013 6mpk and Hu303-005 6 mpk bothhave certain anti-tumor effect, and the tumor inhibition rates were27.25% (p<0.05) and 34.94% (p<0.01), respectively. There was asignificant difference compared to control group (p<0.001 vs hIGg).

TABLE 11 Effect of humanized anti-LAG-3 antibody on subcutaneouslyinoculated U-87MG Tumor in Mice. Day 0 Day 14 % TGI at Groups Dose(mpk)Mean ± SEM (mm³) Mean ± SEM(mm³) P(vs hIgG) Day 14 hIgG control 6 37.9 ±2.6 247.1 ± 26.5  — — Hu229-013 6 37.9 ± 2.5 190.1 ± 26.2* <0.05 27.25%Hu303-005 6 37.7 ± 2.4  173.5 ± 26.5** <0.01 34.94% D 0: First time ofadministration; *p < 0.05, **p < 0.01, ***p < 0.001 vs hIGg by two wayANOVA.

Test Example 7 Pharmacokinetics (PK) Assay of Humanized Anti-LAG-3Antibodies Hu229-013 and Hu303-005 in Mouse

Eighteen ICR mice, male, weighing from 18 to 22 g, were purchased fromthe West Poole-Bikai Experimental Animal Co., Ltd. During the feedingperiod, the mice were given ad libitum access to water and diet, theduration for adaption in the laboratory environment is not less than 3days, with 12/12 hour light/dark cycle regulation, at the temperature of16-26° C. and relative humidity of 40-70%. ICR mice were numbered andrandomly divided into different groups one day before starting theexperiment, each group of 3 mice. On the day of the experiment, twogroups of mice were injected intravenously with humanized candidateantibody (Hu229-013) at dose of 3 mg/kg and 10 mg/kg, respectively; Theother two groups of mice were injected intravenously with humanizedcandidate antibody (Hu303-005) at dose of 3 mg/kg and 10 mg/kg,respectively. The volume for intravenous injection is of 20 ml/kg.

The blood samples were collected at time point of 15 min, 8 h, 1 d, 2 d,7 d, 10 d, 14 d, 21 d, 28 d, and 35 d post administration. Each timeabout 0.1 ml of whole blood was taken into the centrifuge tube withoutanticoagulant, placed at 4° C. for 30 min, and then centrifuged at 1000g for 15 min. After that, the supernatant was pipetted into the EP tubeand stored at −80° C.

The concentration of drug in serum was measured by ELISA, and the T1/2and other main parameters were calculated by Winnolin software. The mainpharmacokinetic parameters are shown in Table 12:

TABLE 12 Pharmacokinetic parameters of Hu229-013 and Hu303-005 in miceHu229-013 Hu303-005 Dose(mg/kg) 3 mg/kg 10 mg/kg 3 mg/kg 10 mg/kgt_(max)(hour) 0.25 0.25 0.25 0.25 C_(max)(ug/ml) 51.6 ± 1.2  130 ± 20.268.2 ± 8.4 243. ± 19.9 AUC_(0-t) (ug/ml*h) 5556 ± 891 17120 ± 4177  6386± 453 22609 ± 1567  AUC_(0-∞) (ug/ml*h)  5871 ± 1036 19736 ± 6142  7124± 581 27061 ± 5154  t_(1/2)(h) 183 ± 54 276 ± 193 232 ± 24 330 ± 194CLz/F(ml/min/kg)  0.0087 ± 0.0015 0.0092 ± 0.0034  0.007 ± 0.0006 0.0063± 0.0011 Vz/F(ml/kg) 134 ± 16 186 ± 107 141 ± 14 168 ± 66  MRT_(0-∞) (h)241 ± 59 353 ± 191 324 ± 37 411 ± 181

The AUCs of humanized LAG-3 antibodies Hu229-013 and Hu303-005 in micewere similar, and the AUCs and peak concentrations of these twoantibodies at the dose of 3 and 10 mg/kg were linearly correlated withthe increasing dose, and showed linear pharmacokinetic characteristic.

Test Example 8 Physical Stability of the Antibody

This test example was used to detect the physical stability of thehumanized anti-LAG-3 antibodies Hu229-013 and Hu303-005.

The thermal stability of different antibodies was detected by DSC(Differential scanning calorimetry), and the stability of antibody indifferent buffer systems and different pH conditions was compared. Thebuffer systems corresponding to different pH values are, for example,PBS (pH7.4), 10 mM His/135 mM NaCl (pH6.0), 10 mM Acetate/135 mM NaCl(pH5.2).

The sample was dissolved in the corresponding buffer, and theconcentration of the sample was controlled at about 1 mg/ml, and wasdetected using MicroCal* VP-Capillary DSC (Malvern). Prior to the test,each sample and blank buffer were degassed with vacuum degassing devicefor 1 to 2 min. 400 μl of sample or blank buffer was added to each well(the loading quantity was 300 μl). Finally, the two pairs of wells wereadded with 14% Decon 90 and ddH₂O, respectively, for washing. After theplate was loaded with sample, the plate was sealed with a plastic cover.Scanning was started from the temperature of 25° C. to 100° C. and thescanning rate is 60° C./h. The results are shown in table 13, indicatingthat both antibodies Hu229-013 and Hu303-005 showed excellent thermalstability in several test systems.

TABLE 13 Sample Buffer Tm-onset (° C.) TM (° C.) Hu229-013 pH 7.4 61.6771.68 pH 6.0 61.96 72.84 pH 5.2 67.3 73.14 Hu303-005 pH 7.4 61.85 70.19pH 6.0 61.01 71.06 pH 5.2 60.84 70.45

The purity of the samples was monitored by SEC-HPLC, and the periodicstability under certain conditions was investigated. Exemplaryconditions are, for example, controlling the concentration of the sampleat about 50 mg/ml and comparing the stability of the differentantibodies which were repeatedly frozen and thawed at −80° C. for 5times in PBS (pH 7.4) system and stored at 4° C. and 40° C. for onemonth. The Xbridge protein BEH SEC 200A (Waters) HPLC column was usedfor detection. The results are shown in Table 14, indicating that bothantibodies showed better stability.

TABLE 14 Hu229-013(Δ %) Hu303-005(Δ %)  4° C. 0.2% 0.2%  40° C. 2.2%2.7% −80° C.  4%  5% frozen-thawed NOTE: Δ % indicates that the rate ofthe reduced HPLC purity

Test Example 9 Chemical Stability of the Antibody

Deamidation is a common chemical modification of antibodies that mayaffect the stability at later stage, especially, it is generallydesirable to avoid high degree of deamidation modification in amino acidresidues at the CDR region or try to reduce the mutations. 500 μg of thetest antibody was dissolved in 500 μl of PBS at pH 7.4, and then wasplaced in water-bath at 40° C. Samples were taken at day 0, 3, 7 for theenzymatic assay. 100 μg of each sample which was taken at different timepoints was dissolved in 100 μl of 0.2 M His-HCl, 8 M Gμa-HCl solution,pH 6.0, and 3 μl of 0.1 g/ml of DTT was added, and then the sample wasplaced in water-bath at 50° C. for 1 h. After ultrafiltration twice with0.02 M His-HCl, pH 6.0, 3 μl of 0.25 mg/mL trypsin was added forenzymatic digestion in water-bath at 37° C. overnight. The deamidationmodification was examined using LC-MS (Agilent 6530 Q-TOF), and theresults are shown in Table 15.

TABLE 15 Light/Heavy Modification Sample chain point Day 0 Day 7Hu229-013 Light chain N28 1.22% 1.27% N53 0.83% 2.91% Hu303-005 Heavychain N55 2.86% 3.55% Light chain N34 2.1% 1.93% NOTE: N representsmodified asparagine which was detected, the number represents theposition counted from the N-terminus of the light chain or heavy chain.Percentage represents the ratio of the deamidation modification to thetotal peptide signal at this site detected by LC-MS.

The results of mass spectrometry show that there was no obvious highproportion of deamidation modification sites in the detected antibodies,suggesting that chemical stability of the antibody is good at laterstage.

1. A LAG-3 antibody or the antigen-binding fragment thereof, comprisingany one or more CDR region sequences selected from the following (i) or(ii), or the amino acid sequences with at least 85% identity to thefollows: (i) HCDR regions as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11; and LCDR regions as shown in SEQ ID NO: 15, SEQ ID NO: 16 andSEQ ID NO: 17; or (ii) HCDR regions as shown in SEQ ID NO: 12, SEQ IDNO: 13 and SEQ ID NO: 14; and LCDR regions as shown in SEQ ID NO: 18,SEQ ID NO: 19 and SEQ ID NO:
 20. 2. The LAG-3 antibody or theantigen-binding fragment thereof according to claim 1, wherein theantibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 asshown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO: 11, respectively, orthe amino acid sequences with at least 85% identity to these sequences;or the antibody heavy chain variable region comprises HCDR1, HCDR2 andHCDR3 as shown in SEQ ID NO: 12,SEQ ID NO: 13 and SEQ ID NO: 14,respectively, or the amino acid sequences with at least 85% identity tothese sequences.
 3. The LAG-3 antibody or the antigen-binding fragmentthereof according to claim 1, wherein the antibody light chain variableregion comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQID NO: 16 and SEQ ID NO: 17, respectively, or the amino acid sequenceswith at least 85% identity to these sequences; or the antibody lightchain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or the aminoacid sequences with at least 85% identity to these sequences.
 4. TheLAG-3 antibody or the antigen-binding fragment thereof according toclaim 1, wherein the antibody or the antigen-binding fragment thereof isa murine antibody or the fragment thereof.
 5. The LAG-3 antibody or theantigen-binding fragment thereof according to claim 1, wherein theantibody light chain variable region further comprises light chain FRregion derived from murine κ chain or a variant thereof, or light chainFR region derived from murine λ chain or a variant thereof; wherein theantibody heavy chain variable region further comprises heavy chain FRregion derived from murine IgG1 or a variant thereof, or heavy chain FRregion derived from murine IgG2 or a variant thereof, or heavy chain FRregion derived from murine IgG3 or a variant thereof.
 6. The LAG-3antibody or the antigen-binding fragment thereof according to claim 4,wherein the murine antibody comprises a heavy chain variable region asshown in SEQ ID NO: 5 and a light chain variable region as shown in SEQID NO: 6, or comprises a heavy chain variable region as shown in SEQ IDNO: 7 and a light chain variable region as shown in SEQ ID NO:
 8. 7. TheLAG-3 antibody or the antigen-binding fragment thereof according toclaim 1, wherein the antibody light chain further comprises light chainconstant region derived from murine κ chain or a variant thereof, orlight chain constant region derived from murine λ chain or a variantthereof; wherein the antibody heavy chain further comprises heavy chainconstant region derived from murine IgG1 or a variant thereof, or heavychain constant region derived from murine IgG2 or a variant thereof, orheavy chain constant region derived from murine IgG3 or a variantthereof.
 8. The LAG-3 antibody or the antigen-binding fragment thereofaccording to claim 1, wherein the antibody or the antigen-bindingfragment thereof is a chimeric antibody or the antigen-binding fragmentthereof.
 9. The LAG-3 antibody or the antigen-binding fragment thereofaccording to claim 1, wherein the antibody or the antigen-bindingfragment thereof is a humanized antibody or the antigen-binding fragmentthereof.
 10. The LAG-3 antibody or the antigen-binding fragment thereofaccording to claim 9, wherein the heavy chain FR sequence of thehumanized antibody heavy chain variable region is derived from acombination sequence of human germline heavy chains IGHV7-4-1*02 andhjh6.1, or a mutant sequence thereof, preferably comprises FR1, FR2, FR3from IGHV7-4-1*02 and FR4 from hjh6.1 or a mutant sequence thereof;alternatively, the heavy chain FR sequence of the humanized antibodyheavy chain variable region is derived from a combination sequence ofhuman germline heavy chain IGHV1-3*01 and hjh6.1, or a mutant sequencethereof, preferably comprises FR1, FR2, FR3 from IGHV1-3*01 and FR4 fromhjh6.1 or a mutant sequence thereof.
 11. The LAG-3 antibody or theantigen-binding fragment thereof according to claim 10, wherein theheavy chain variable region sequence of the humanized antibody is asshown in SEQ ID NO: 21 or SEQ ID NO: 29, or has at least 85% identity tothese amino acid sequences; preferably has 1-10 amino acid changes inthe heavy chain variable region.
 12. The LAG-3 antibody orantigen-binding fragment thereof according to claim 10, wherein theheavy chain FR sequence of the humanized antibody has 0-10 amino acidback-mutations; preferably, the back-mutation is present on heavy chainFR region of the combination sequence of human germline heavy chainsIGHV7-4-1*02 and hjh6.1, and is one or more amino acid back-mutationsselected from the group consisting of E46K, R38K, V93T and Y95F; or,preferably, the back-mutation is present on heavy chain FR region of thecombination sequence of human germline heavy chains IGHV1-3*01 andhjh6.1, and is one or more amino acid back-mutations selected from thegroup consisting of F29L, A97T, M48I, V68A, 170L, R72V and T74K.
 13. TheLAG-3 antibody or the antigen-binding fragment thereof according toclaim 10, wherein the heavy chain variable region sequence of thehumanized antibody is selected from the group consisting of SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO: 25, or the sequenceswith at least 85% identity to these sequences; or selected from thegroup consisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32 and SEQ IDNO: 33, or the sequences with at least 85% identity to these sequences.14. The LAG-3 antibody or the antigen-binding fragment thereof accordingto claim 9, wherein the light chain FR sequence of the humanizedantibody light chain variable region is derived from a combinationsequence of human germline light chain IGKV1-39*01 and hjk4.1, or amutant sequence thereof and comprises FR1, FR2, FR3 from human germlinelight chain IGHV1-39*01 and FR4 from hjh4.1 or a mutant sequencethereof.
 15. The LAG-3 antibody or the antigen-binding fragment thereofaccording to claim 14, wherein the light chain variable region sequenceof the humanized antibody is as shown in SEQ ID NO: 22 or SEQ ID NO: 30,or the sequence with at least 85% identity to these sequences;preferably has 1-10 amino acid changes on the light chain variableregion.
 16. The LAG-3 antibody or the antigen-binding fragment thereofaccording to claim 14, wherein the light chain FR sequence of thehumanized antibody has 0-10 amino acid back-mutations, preferably one ormore back-mutations selected from the group consisting of D70Q, F71Y,I48V and A43S, or preferably one or more back-mutations selected fromthe group consisting of L46R, G66R, S60K, P44F, Y36L, K42G, I21L andT85D.
 17. The LAG-3 antibody or the antigen-binding fragment thereofaccording to claim 14, wherein the light chain variable region sequenceof the humanized antibody is selected from the group consisting of SEQID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 or thesequence with at least 85% identity to these sequences; or is selectedfrom the group consisting of SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36 and SEQ ID NO: 37, or the sequence with at least 85%identity to these sequences.
 18. The LAG-3 antibody or theantigen-binding fragment thereof according to claim 9, wherein thehumanized antibody comprising: (a) heavy chain variable region, whereinthe heavy chain variable region sequence is selected from the groupconsisting of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO:25, or the sequence with at least 85% identity to these sequences;alternatively, the heavy chain variable region sequence is selected fromthe group consisting of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 andSEQ ID NO: 33, or the sequence with at least 85% identity to thesesequences; and/or (b) light chain variable region, wherein the lightchain variable region sequence is selected from the group consisting ofSEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, or thesequence with at least 85% identity to these sequences; alternatively,the light chain variable region sequence is selected from the groupconsisting of sequence SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQID NO: 36 and SEQ ID NO: 37, or the sequence with at least 85% identityto these sequences.
 19. The LAG-3 antibody or the antigen-bindingfragment thereof according to claim 9, wherein the antibody comprising acombination of a heavy chain variable region and a light chain variableregion selected from the group consisting of: 1) the heavy chainvariable region of SEQ ID NO: 21 and the light chain variable region ofSEQ ID NO: 22; 2) the heavy chain variable region of SEQ ID NO: 21 andthe light chain variable region of SEQ ID NO: 26; 3) the heavy chainvariable region of SEQ ID NO: 21 and the light chain variable region ofSEQ ID NO: 27; 4) the heavy chain variable region of SEQ ID NO: 21 andthe light chain variable region of SEQ ID NO: 28; 5) the heavy chainvariable region of SEQ ID NO: 23 and the light chain variable region ofSEQ ID NO: 22; 6) the heavy chain variable region of SEQ ID NO: 23 andthe light chain variable region of SEQ ID NO: 26; 7) the heavy chainvariable region of SEQ ID NO: 23 and the light chain variable region ofSEQ ID NO: 27; 8) the heavy chain variable region of SEQ ID NO: 23 andthe light chain variable region of SEQ ID NO: 28; 9) the heavy chainvariable region of SEQ ID NO: 24 and the light chain variable region ofSEQ ID NO: 22; 10) the heavy chain variable region of SEQ ID NO: 24 andthe light chain variable region of SEQ ID NO: 26; 11) the heavy chainvariable region of SEQ ID NO: 24 and the light chain variable region ofSEQ ID NO: 27; 12) the heavy chain variable region of SEQ ID NO: 24 andthe light chain variable region of SEQ ID NO: 28; 13) the heavy chainvariable region of SEQ ID NO: 25 and the light chain variable region ofSEQ ID NO: 22; 14) the heavy chain variable region of SEQ ID NO: 25 andthe light chain variable region of SEQ ID NO: 26; 15) the heavy chainvariable region of SEQ ID NO: 25 and the light chain variable region ofSEQ ID NO: 27; 16) the heavy chain variable region of SEQ ID NO: 25 andthe light chain variable region of SEQ ID NO: 28; 17) the heavy chainvariable region of SEQ ID NO: 29 and the light chain variable region ofSEQ ID NO: 30; 18) the heavy chain variable region of SEQ ID NO: 29 andthe light chain variable region of SEQ ID NO: 34; 19) the heavy chainvariable region of SEQ ID NO: 29 and the light chain variable region ofSEQ ID NO: 35; 20) the heavy chain variable region of SEQ ID NO: 29 andthe light chain variable region of SEQ ID NO: 36; 21) the heavy chainvariable region of SEQ ID NO: 29 and the light chain variable region ofSEQ ID NO: 37; 22) the heavy chain variable region of SEQ ID NO: 31 andthe light chain variable region of SEQ ID NO: 30; 23) the heavy chainvariable region of SEQ ID NO: 31 and the light chain variable region ofSEQ ID NO: 34; 24) the heavy chain variable region of SEQ ID NO: 31 andthe light chain variable region of SEQ ID NO: 35; 25) the heavy chainvariable region of SEQ ID NO: 31 and the light chain variable region ofSEQ ID NO: 36; 26) the heavy chain variable region of SEQ ID NO: 31 andthe light chain variable region of SEQ ID NO: 37; 27) the heavy chainvariable region of SEQ ID NO: 32 and the light chain variable region ofSEQ ID NO: 30; 28) the heavy chain variable region of SEQ ID NO: 32 andthe light chain variable region of SEQ ID NO: 34; 29) the heavy chainvariable region of SEQ ID NO: 32 and the light chain variable region ofSEQ ID NO: 35; 30) the heavy chain variable region of SEQ ID NO: 32 andthe light chain variable region of SEQ ID NO: 36; 31) the heavy chainvariable region of SEQ ID NO: 32 and the light chain variable region ofSEQ ID NO: 37; 32) the heavy chain variable region of SEQ ID NO: 33 andthe light chain variable region of SEQ ID NO: 30; 33) the heavy chainvariable region of SEQ ID NO: 33 and the light chain variable region ofSEQ ID NO: 34; 34) the heavy chain variable region of SEQ ID NO: 33 andthe light chain variable region of SEQ ID NO: 35; 35) the heavy chainvariable region of SEQ ID NO: 33 and the light chain variable region ofSEQ ID NO: 36; and 36) the heavy chain variable region of SEQ ID NO: 33and the light chain variable region of SEQ ID NO:
 37. 20. The LAG-3antibody or the antigen-binding fragment thereof according to any one ofclaims 7-15, wherein the heavy chain of the chimeric antibody or thehumanized antibody further comprises a heavy chain constant regionderived from human IgG1, IgG2, IgG3 or IgG4 or a variant thereof,preferably a heavy chain constant region derived from human IgG4 or avariant thereof, most preferably a heavy chain constant region as shownin SEQ ID NO: 38; wherein the light chain of the chimeric antibody orthe humanized antibody further comprises a light chain constant regionderived from human κ chain, human λ chain or a variant thereof, mostpreferably a light chain constant region as shown in SEQ ID NO:
 39. 21.An isolated monoclonal antibody or antigen-binding fragment thereof thatcompetes for binding to LAG-3 with the monoclonal antibody orantigen-binding fragment thereof according to any one of claims 1-20.22. A pharmaceutical composition, comprising therapeutically effectiveamount of the LAG-3 antibody or the antigen-binging fragment thereofaccording to any one of claims 1-21 and one or more pharmaceuticallyacceptable carriers, diluents or excipients.
 23. An isolated nucleicacid encoding the LAG-3 antibody or the antigen-binding fragment thereofaccording to any one of claims 1 to
 21. 24. An expression vectorcomprising the nucleic acid according to claim
 23. 25. A host celltransformed with the expression vector according to claim 24, whereinthe host cell is selected from the group consisting of prokaryotic cellsand eukaryotic cells, preferably eukaryotic cells, more preferablymammalian cells.
 26. A method for preparing a LAG-3 antibody or theantigen-binding fragment thereof, wherein the method comprisesexpressing the antibody or the antigen-binding fragment thereof in thehost cell of claim 25 and isolating the antibody or the antigen-bindingfragment thereof from the host cell.
 27. A method for inhibiting thegrowth of tumor cells in a subject, wherein the method comprisesadministering to the subject therapeutically effective amount of theLAG-3 antibody or the antigen-binding fragment thereof according to anyone of claims 1 to 21, or administering to the subject thepharmaceutical composition according to claim 22 or the nucleic acidaccording to claim 23, thereby inhibiting tumor growth in the subject.28. The use of the LAG-3 antibody or the antigen-binding fragmentaccording to any one of claims 1-21, or the pharmaceutical compositionaccording to claim 22 or the nucleic acid according to claim 23, in thepreparation of a medicament for treatment of a disease or a conditionassociated with the involvement of pathogenic T cells, wherein thedisease or the condition is preferably cancer; the cancer includes butnot limited to ovarian cancer, melanoma, prostate cancer, intestinalcancer, stomach cancer, esophageal cancer, breast cancer, lung cancer,renal cancer, pancreatic cancer, uterine cancer, liver cancer, bladdercancer, cervical cancer, oral cancer, brain cancer, testicular cancer,skin cancer, thyroid cancer, and hematological malignant tumorsincluding myeloma and chronic and acute leukemia.